Method of transferring at least two saccharide units with a polyglycosyltransferase, a polyglycosyltransferase and gene encoding a polyglycosyltransferase

ABSTRACT

The present invention relates to a method of transferring at least two saccharide units with a polyglycosyltransferase, a polyglycosyltransferase and a gene encoding such a polyglycosyltransferase.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of transferring at least twosaccharide units with a polyglycosyltransferase, apolyglycosyltransferase and a gene encoding such apolyglycosyltransferase.

2. Discussion of the Background

Biosynthesis of Oligosaccharides

Oligosaccharides are polymers of varying number of residues, linkages,and subunits. The basic subunit is a carbohydrate monosaccharide orsugar, such as mannose, glucose, galactose, N-acetylglucosamine,N-acetylgalactosamine, and the like. The number of different possiblestereoisomeric oligosaccharide chains is enormous.

Oligosaccharides and polysaccharides play an important role in proteinfunction and activity, by serving as half-life modulators, and, in someinstances, by providing structure. Oligosaccharides are critical to theantigenic variability, and hence immune evasion, of Neisseria,especially gonococcus.

Numerous classical techniques for the synthesis of carbohydrates havebeen developed, but these techniques suffer the difficulty of requiringselective protection and deprotection. Organic synthesis ofoligosaccharides is further hampered by the lability of many glycosidicbonds, difficulties in achieving regioselective sugar coupling, andgenerally low synthetic yields. In short, unlike the experience withpeptide synthesis, traditional synthetic organic chemistry cannotprovide for quantitative, reliable synthesis of even fairly simpleoligosaccharides.

Recent advances in oligosaccharide synthesis have occurred with theisolation of glycosyltransferases from natural sources. These enzymescan be used in vitro to prepare oligosaccharides and polysaccharides(see, e.g., Roth, U.S. Pat. No. 5,180,674). The advantage ofbiosynthesis with glycosyltransferases is that the glycosidic linkagesformed by enzymes are highly stereo and regiospecific. However, eachenzyme catalyzes linkage of specific sugar donor residues to otherspecific acceptor molecules, e.g., an oligosaccharide or lipid. Thus,synthesis of a desired oligosaccharide has required the use of adifferent glycosyltransferase for each different saccharide unit beingtransferred.

More specifically, such glycosyltransferases have only provided for thetransfer of a single saccharide unit, specific for theglycosyltransferase. For example, a galactosyltransferase would transferonly galactose, a glucosyltransferase would transfer only glucose, anN-acetylglucosaminlytransferase would transfer only N-acetylglucosamineand a sialyl transferase would transfer only sialic acid.

However, the lack of generality of glycosyltransferases makes itnecessary to use a different glycosyltransferase for every differentsugar donor being transferred. As the usefulness of oligosaccharidecompounds expands, the ability to transfer more than one sugar donorwould provide a tremendous advantage, by decreasing the number ofglycosyltransferases necessary to form necessary glycosidic bonds.

In addition, a glycosyltransferase which transferred at least twodifferent sugar donors would be advantageous in synthesizing twoglycosidic bonds of at least a trisaccharide, using the sameglycosyltransferase.

A locus involved in the biosynthesis of gonococcal lipooligosaccharide(LOS) has been reported as being cloned from the gonococcal strain F62(Gotschlich, J. Exp. Med. (1994) 180, 2181-2190). Five genes lgtA, lgtB,lgtC, lgtD and lgtE are reported, and based on deletion experiments,activities are postulated, as encoding for glycosyltransferases. Due tothe uncertainty caused by the nature of the deletion experiments, theexact activity of the proteins encoded by each of the genes was notascertained and some of the genes are only suggested as beingresponsible for one or another activity, in the alternative. The genelgtA is suggested as most likely to code for a GlcNAc transferase.

The transfer of more than one different saccharide moiety, by apolyglycosyltransferase has heretofore been unreported.

SUMMARY OF THE INVENTION

The present invention is directed to a method of transferring at leasttwo saccharide units with a polyglycosyltransferase, apolyglycosyltransferase and nucleic acids encoding apolyglycosyltransferase.

Accordingly, in one aspect, the invention is directed to a method oftransferring at least two saccharide units with apolyglycosyltransferase.

Accordingly, another aspect of the invention is directed to a method oftransferring at least two saccharide units with apolyglycosyltransferase, which transfers both GlcNAc and GalNAc from thecorresponding sugar nucleotides to a sugar acceptor.

According to another aspect of the invention, a polyglycosyltransferaseis obtained from a bacteria of the genus Neisseria, Escherichia orPseudomonas.

According aspect of the invention, is directed to a method of making atleast two oligosaccharide compounds, from the same acceptor, with apolyglycosyltransferase.

Another, another aspect of the invention is directed to a method ofmaking at least two oligosaccharide compounds from the same acceptorwith a polyglycosyltransferase which transfers both GlcNAc and GalNAc,from the corresponding sugar nucleotides, to the sugar acceptor.

Another embodiment of the present invention is directed to a method oftransferring an N-acetylgalactosamine using a glycosyltransferase of SEQID NO: 8.

In specific embodiments, the invention relates to a nucleic acid thathas a nucleotide sequence which encodes for the polypeptide sequenceshown in SEQ ID NO: 8.

The functionally active polyglycosyltransferase of the invention ischaracterized by catalyzing both the addition of GalNAc β1→3 to Gal andthe addition of GlcNAc β1→3 to Gal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: provides the amino acid sequence of a polyglycosyltransferase ofSEQ ID NO: 3.

FIGS. 2A -2H: provides the polynucleotide sequence of a LOS encodinggene isolated from N. gonorrhoeae (SEQ ID NO:1), of which nucleotides445-1488 encode for a polyglycosyltransferase.

DETAILED DESCRIPTION OF THE INVENTION

As disclosed above, the present invention provides for a method oftransferring at least two saccharide units with apolyglycosyltransferase, a gene encoding for a polyglycosyltransferase,and a polyglycosyltransferase. The polyglycosyltransferases of theinvention can be used for in vitro biosynthesis of variousoligosaccharides, such as the core oligosaccharide of the human bloodgroup antigens, i.e., lacto-N-neotetraose.

Cloning and expression of a polyglycosyltransferase of the invention canbe accomplished using standard techniques, as disclosed herein. Such apolyglycosyltransferase is useful for biosynthesis of oligosaccharidesin vitro, or alternatively genes encoding such a polyglycosyltransferasecan be transfected into cells, e.g., yeast cells or eukaryotic cells, toprovide for alternative glycosylation of proteins and lipids.

The instant invention is based, in part, on the discovery that apolyglycosyltransferase isolated from Neisseria gonorrhoeae is capableof transferring both GlcNAc β1-3 to Gal and GalNAc β1-3 to Gal, from thecorresponding sugar nucleotides.

An operon encoding fire proteins having glycosyltransferase activity, isreported by Gotschlich, U.S. Pat. No. 5,545,553, by cloning of a locusinvolved in the biosynthesis of gonococcal LOS, strain F62. The proteinsequence identified herein as SEQ ID NO: 8, a 348 amino acid protein,has now been discovered to have a polyglycosyltransferase activity. Morespecifically, the protein sequence identified as SEQ ID NO: 8 has beendiscovered to transfer both GlcNAc β1-3 to Gal and GalNAc β1-3 to Gal,from the corresponding sugar nucleotides.

In addition to the protein sequence SEQ ID NO: 8 the nucleotide sequenceencoding this protein sequence reported in U.S. Pat. No. 5,545,553, anew polyglycosyltransferase has been discovered which transfers twodifferent sugar units. This protein is similar to the protein of SEQ ID:8, with the deletion of one or two of the five glycine units occurringbetween amino acid nos 86-90 of lgtA. In addition, it has beendetermined that an additional amino acid sequence-Tyr-Ser-Arg-Asp-Ser-Ser (SEQ ID NO: 7), can be appended to the carboxyterminus of Ile (amino acid no 348) of SEQ ID NO: 3, while retaining thepolyglycosyltransferase activity.

A polynucleotide sequence encoding for a polyglycosyltransferase issimilar to the sequence of nucleotides 445 to 1488 of an LOS isolatedfrom N. gonorrhoeae (see FIG. 2 (SEQ ID NO:1) in which three or six ofthe guanine units occuring between nucleic acids 700 to 715 have beendeleted.

Another polynucleotide sequence is similar to the sequence ofnucleotides 445 to 1488 of an LOS isolated from N. gonorrhoeae (see FIG.2), in which nucleic acids sufficient to encode the amino acid sequence-Tyr-Ser-Arg-Asp-Ser-Ser (SEQ ID NO:7), can be appended to nucleic acid1488 and the protein encoded by the nucleotide sequence (nucleotides 445to 1488 and the appended sequence) retains polyglycosyltransferaseactivity.

Abbreviations used throughout this specification include:Lipopolysaccharide, LPS; Lipooligosaccharide, LOS; N-Acetyl-neuraminicacid cytidine mono phosphate, CMP-NANA; wild type, wt; Gal, galactose;Glc, glucose; NAc, N-acetyl (e.g., GalNAc or GlcNAc).

Isolation of Genes for Polyglycosyltransferases

Any Neisseria bacterial cell can potentially serve as the nucleic acidsource for the molecular cloning of a polyglycosyltransferase gene. In aspecific embodiment, infra, the genes are isolated from Neisseriagonorrhoeae. The DNA may be obtained by standard procedures known in theart from cloned DNA (e.g., a DNA "library"), by chemical synthesis, bycDNA cloning, or by the cloning of genomic DNA, or fragments thereof,purified from the desired cell (See, for example, Sambrook et al., 1989,supra; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRLPress, Ltd., Oxford, U.K. Vol. 1, II). For example, a N. gonorrhoeaegenomic DNA can be digested with a restriction endonuclease orendonucleases, e.g., Sau3A, into a phage vector digested with arestriction endonuclease or endonucleases, e.g., BamHI/EcoRI, forcreation of a phage genomic library. Whatever the source, the geneshould be molecularly cloned into a suitable vector for propagation ofthe gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired polyglycosyltransferase gene may beaccomplished in a number of ways. For example, the generated DNAfragments may be screened by nucleic acid hybridization to the labeledprobe synthesized with a sequence as disclosed herein (Benton and Davis,1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad.Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology tothe probe will hybridize.

Suitable probes can be generated by PCR using random primers. Inparticular a probe which will hybridize to the polynucleotide sequenceencoding for a four or five glycine residue (i.e., a twelve or fifteenguanine residue) would be a suitable probe for apolyglycosyltransferase.

The presence of the polyglycosyltransferase gene may be detected byassays on the physical, chemical, or immunological properties of itsexpressed product. For example, these assays may screen for DNA clonesthat produce a protein that, e.g., has similar or identicalelectrophoretic migration, isoelectric focusing behavior, proteolyticdigestion maps, and proteolytic activity, or functional properties, suchas polyglycosyltransferase activity, the ability of apolyglycosyltransferase protein to mediate transfer of two differentsaccharide units to an acceptor molecule.

Alternatives to isolating a polyglycosyltransferase genomic DNA include,but are not limited to, chemically synthesizing the gene sequence itselffrom a known sequence that encodes a polyglycosyltransferase. In anotherembodiment, DNA for a polyglycosyltransferase gene can be isolated byPCR using oligonucleotide primers designed from the nucleotide sequencesdisclosed herein. Other methods are possible and within the scope of theinvention.

The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. In a specific aspect of theinvention, the polyglycosyltransferase coding sequence is inserted in anE. coli cloning vector. Other examples of vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors,pmal-c, pFLAG, etc. The insertion into a cloning vector can, forexample, be accomplished by ligating the DNA fragment into a cloningvector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In a specific embodiment, PCR primers containing such linkersites can be used to amplify the DNA for cloning. Recombinant moleculescan be introduced into host cells via transformation, transfection,infection, electroporation, etc., so that many copies of the genesequence are generated.

Transformation of host cells with recombinant DNA molecules thatincorporate the isolated polyglycosyltransferase gene or synthesized DNAsequence enables generation of multiple copies of the gene. Thus, thegene may be obtained in large quantities by growing transformants,isolating the recombinant DNA molecules from the transformants and, whennecessary, retrieving the inserted gene from the isolated recombinantDNA.

The present invention also relates to vectors containing genes encodingtruncated forms of the enzyme (fragments) and derivatives ofpolyglycosyltransferases that have the same functional activity as apolyglycosyltransferase. The production and use of fragments andderivatives related to polyglycosyltransferases are within the scope ofthe present invention. In a specific embodiment, the fragment orderivative is functionally active, i.e., capable of mediating transferof two different sugar donors to acceptor moieties.

Truncated fragments of the polyglycosyltransferases can be prepared byeliminating N-terminal, C-terminal, or internal regions of the proteinthat are not required for functional activity. Usually, such portionsthat are eliminated will include only a few, e.g., between 1 and 5,amino acid residues, but larger segments may be removed.

Chimeric molecules, e.g., fusion proteins, containing all or afunctionally active portion of a polyglycosyltransferase of theinvention joined to another protein are also envisioned. Apolyglycosyltransferase fusion protein comprises at least a functionallyactive portion of a non-glycosyltransferase protein joined via a peptidebond to at least a functionally active portion of apolyglycosyltransferase polypeptide. The non-glycosyltransferasesequences can be amino- or carboxy-terminal to thepolyglycosyltransferase sequences. Expression of a fusion protein canresult in an enzymatically inactive polyglycosyltransferase fusionprotein. A recombinant DNA molecule encoding such a fusion proteincomprises a sequence encoding at least a functionally active portion ofa non-glycosyltransferase protein joined in-frame to thepolyglycosyltransferase coding sequence, and preferably encodes acleavage site for a specific protease, e.g., thrombin or Factor Xa,preferably at the polyglycosyltransferase-non-glycosyltransferasejuncture. In a specific embodiment, the fusion protein may be expressedin Escherichia coli.

In particular, polyglycosyltransferase derivatives can be made byaltering encoding nucleic acid sequences by substitutions, additions ordeletions that provide for functionally equivalent molecules. Due to thedegeneracy of nucleotide coding sequences, other DNA sequences whichencode substantially the same amino acid sequence as apolyglycosyltransferase gene may be used in the practice of the presentinvention. These include but are not limited to nucleotide sequencescomprising all or portions of polyglycosyltransferase genes that arealtered by the substitution of different codons that encode the sameamino acid residue within the sequence, thus producing a silent change.Likewise, the polyglycosyltransferase derivatives of the inventioninclude, but are not limited to, those containing, as a primary aminoacid sequence, all or part of the amino acid sequence of apolyglycosyltransferase including altered sequences in whichfunctionally equivalent amino acid residues are substituted for residueswithin the sequence resulting in a conservative amino acid substitution.For example, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity, which acts as afunctional equivalent, resulting in a silent alteration. Substitutes foran amino acid within the sequence may be selected from other members ofthe class to which the amino acid belongs. For example, the nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

The genes encoding polyglycosyltransferase derivatives and analogs ofthe invention can be produced by various methods known in the art (e.g.,Sambrook et al., 1989, supra). The sequence can be cleaved atappropriate sites with restriction endonuclease(s), followed by furtherenzymatic modification if desired, isolated, and ligated in vitro. Inthe production of the gene encoding a derivative or analog ofpolyglycosyltransferase, care should be taken to ensure that themodified gene remains within the same translational reading frame as thepolyglycosyltransferase gene, uninterrupted by translational stopsignals, in the gene region where the desired activity is encoded.

Additionally, the polyglycosyltransferase nucleic acid sequence can bemutated in vitro or in vivo, to create and/or destroy translation,initiation, and/or termination sequences, or to create variations incoding regions and/or form new restriction endonuclease sites or destroypreexisting ones, to facilitate further in vitro modification. Anytechnique for mutagenesis known in the art can be used, including butnot limited to, in vitro site-directed mutagenesis (Hutchinson, C., etal., 1978, J. Biol. Chem. 253:6551; Zoller and Smith, 1984, DNA3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986,Proc. Natl. Acad. Sci. U.S.A. 83:710), use of TABO linkers (Pharmacia),etc. PCR techniques are preferred for site directed mutagenesis (seeHiguchi, 1989, "Using PCR to Engineer DNA", in PCR Technology:Principles and Applications for DNA Amplification, H. Erlich, ed.,Stockton Press, Chapter 6, pp. 61-70).

While a polyglycosyltransferase has been isolated from a bacteria ofNeisseria gonorrhoeae, polyglycosyltransferases can also be isolatedfrom other bacterial species of Neisseria. Exemplary Neisseria bacterialsources include N. animalis (ATCC 19573), N. canis (ATCC 14687), N.cinerea (ATCC 14685), N. cuniculi (ATCC 14688), N. denitrificans (ATCC14686), N. elongata (ATCC 25295), N. elongata subsp glycolytica (ATCC29315), N. elongata subsp. nitroreducens (ATCC 49377), N. flavescens(ATCC 13115), N. gonorrhoeae (ATCC 33084), N. lactamica (ATCC 23970), N.macaca (ATCC 33926), N. meningitidis, N. mucosa (ATCC 19695), N. mucosasubsp. heidelbergensis (ATCC 25998), N. polysaccharea (ATCC 43768), N.sicca (ATCC 29256) and N. subflava (ATCC 49275). Strains assignedAmerican Type Culture Collection (ATCC) accession numbers are availablefrom the ATCC, 1201 Parklawn Drive, Rockville, Md. 20852. In additionpolyglycosyltransferases can be isolated from Branhamella catarrhalis,Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa andPseudomonas cepacia.

Expression of a Polyglycosyltransferase

The gene coding for a polyglycosyltransferase, or a functionally activefragment or other derivative thereof, can be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. An expression vector also preferably includes areplication origin. The necessary transcriptional and translationalsignals can also be supplied by the native polyglycosyltransferase geneand/or its flanking regions. A variety of host-vector systems may beutilized to express the protein-coding sequence. Preferably, however, abacterial expression system is used to provide for high level expressionof the protein with a higher probability of the native conformation.Potential host-vector systems include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

Preferably, the periplasmic form of the polyglycosyltransferase(containing a signal sequence) is produced for export of the protein tothe Escherichia coli periplasm or in an expression system based onBacillus subtilis.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).

Expression of a nucleic acid sequence encoding a polyglycosyltransferaseor peptide fragment may be regulated by a second nucleic acid sequenceso that the polyglycosyltransferase or peptide is expressed in a hosttransformed with the recombinant DNA molecule. For example, expressionof a polyglycosyltransferase may be controlled by any promoter/enhancerelement known in the art, but these regulatory elements must befunctional in the host selected for expression. For expression inbacteria, bacterial promoters are required. Eukaryotic viral oreukaryotic promoters, including tissue specific promoters, are preferredwhen a vector containing a polyglycosyltransferase gene is injecteddirectly into a subject for transient expression, resulting inheterologous protection against bacterial infection, as described indetail below. Promoters which may be used to controlpolyglycosyltransferase gene expression include, but are not limited to,the SV40 early promoter region (Benoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3' long terminal repeat ofRous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expressionvectors such as the O-lactamase promoter (Villa-Kamaroff, et al., 1978,Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter(DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also"Useful proteins from recombinant bacteria" in Scientific American,1980, 242:74-94; and the like.

Expression vectors containing polyglycosyltransferase gene inserts canbe identified by four general approaches: (a) PCR amplification of thedesired plasmid DNA or specific mRNA, (b) nucleic acid hybridization,(c) presence or absence of "marker" gene functions, and (d) expressionof inserted sequences. In the first approach, the nucleic acids can beamplified by PCR with incorporation of radionucleotides or stained withethidium bromide to provide for detection of the amplified product. Inthe second approach, the presence of a foreign gene inserted in anexpression vector can be detected by nucleic acid hybridization usingprobes comprising sequences that are homologous to an insertedpolyglycosyltransferase gene. In the third approach, the recombinantvector/host system can be identified and selected based upon thepresence or absence of certain "marker" gene functions (e.g.,β-galactosidase activity, PhoA activity, thymidine kinase activity,resistance to antibiotics, transformation phenotype, occlusion bodyformation in baculovirus, etc.) caused by the insertion of foreign genesin the vector. If the polyglycosyltransferase gene is inserted withinthe marker gene sequence of the vector, recombinants containing thepolyglycosyltransferase insert can be identified by the absence of themarker gene function. In the fourth approach, recombinant expressionvectors can be identified by assaying for the activity of thepolyglycosyltransferase gene product expressed by the recombinant. Suchassays can be based, for example, on the physical or functionalproperties of the polyglycosyltransferase gene product in in vitro assaysystems, e.g., polyglycosyltransferase activity. Once a suitable hostsystem and growth conditions are established, recombinant expressionvectors can be propagated and prepared in quantity.

Biosynthesis of Oligosaccharides

The polyglycosyltransferases of the present invention can be used in thebiosynthesis of oligosaccharides. The polyglycosyltransferases of theinvention are capable of stereospecific conjugation of two specificactivated saccharide units to specific acceptor molecules. Suchactivated saccharides generally consist of uridine or guanosinediphosphate and cytidine monophosphate derivatives of the saccharides,in which the nucleoside mono- and diphosphate serves as a leaving group.Thus, the activated saccharide may be a saccharide-UDP, asaccharide-GDP, or a saccharide-CMP. In specific embodiments, theactivated saccharide is UDP-GlcNAC, UDP-GalNAc, or UDP-Gal.

Within the context of the claimed invention, two different saccharideunits means saccharides which differ in structure and/or stereochemistryat a position other than C₁ and accordingly the pyranose and furanose ofthe same carbon backbone are considered to be the same saccharide unit,while glucose and galactose (i.e. C₄ isomers) are considered different.

A glycosyltransferase typically has a catalytic activity of from about 1to 250 turnovers/sec in order to be considered to possess a specificglycosyltransferase activity. Accordingly, each individualglycosyltransferase activity of the polyglycosyltransferase of thepresent invention is within the range of from 1 to 250 turnovers/sec,preferably from 5 to 100 turnovers/sec, more preferably from 10 to 30turnovers/sec.

In addition to absolute glycosyltransferase activity, thepolyglycosyltransferases used according to the methods of the inventioncatalyze a glycidic linkage having a relative activity of from 0.1 to 10times, preferably from 0.2 to 5 times, more preferably from 0.5 to 2times and most preferably from 0.8 to 1.5 times, the rate of any one ofthe other glycosyltransferase activity identified for that particularglycosyltransferase activity identified for that particularglycosyltransferase.

The term "acceptor moiety" as used herein refers to the molecules towhich the polyglycosyltransferase transfers activated sugars.

For the synthesis of an oligosaccharide, a polyglycosyltransferase iscontacted with an appropriate activated saccharide and an appropriateacceptor moiety under conditions effective to transfer and covalentlybond the saccharide to the acceptor molecule. Conditions of time,temperature, and pH appropriate and optimal for a particular saccharineunit transfer can be determined through routine testing; generally,physiological conditions will be acceptable. Certain co-reagents mayalso be desirable; for example, it may be more effective to contact thepolyglycosyltransferase with the activated saccharide and the acceptormoiety in the presence of a divalent cation.

According to the invention, the polyglycosyltransferase enzymes can becovalently or non-covalently immobilized on a solid phase support suchas SEPHADEX, SEPHAROSE, or poly(acrylamide-co-N-acryloxysucciimide)(PAN) resin. A specific reaction can be performed in an isolatedreaction solution, with facile separation of the solid phase enzyme fromthe reaction products. Immobilization of the enzyme also allows for acontinuous biosynthetic stream, with the specificpolyglycosyltransferases attached to a solid support, with the supportsarranged randomly or in distinct zones in the specified order in acolumn, with passage of the reaction solution through the column andelution of the desired oligosaccharide at the end. An efficient methodfor attaching the polyglycosyltransferase to a solid support and usingsuch immobilized polyglycosyltransferases is described in U.S. Pat. No.5,180,674, issued Jan. 19, 1993 to Roth, which is specificallyincorporated herein by reference in its entirety.

An oligosaccharide, e.g., a disaccharide, prepared using apolyglycosyltransferase of the present invention, can serve as anacceptor moiety for further synthesis, either using otherpolyglycosyltransferases of the invention, or glycosyltransferases knownin the art (see, e.g., Roth, U.S. Pat. No. 5,180,674).

Alternatively, the polyglycosyltransferases of the present invention canbe used to prepare GalNAcβ1-3-Galβ1-4-GlcNAcβ-3-Galβ1-4-Glc orGalNAcβ1-3-Galβ1-4-GlcNAcβ1-3-Galβ1from lactose or lactosaminerespectively, in which a polyglycosyltransferase is used to synthesizeboth the GlcNAc β1-3-Gal and GalNAc β1-3 Gal linkages.

Accordingly, a method for preparing an oligosaccharide having thestructure GalNAcβ1-3-Galβ1-4-GlcNAcβ1-3-Galβ1-4-Glc comprisessequentially performing the steps of:

a) contacting a reaction mixture comprising an activated GlcNAc (such asUDP-GlcNAc) to lactose with a polyglycosyltransferase having an aminoacid sequence of SEQ ID NO:3, or a functionally active fragment thereof;

b) contacting a reaction mixture comprising an activated Gal (i.eUDP-Gal) to the acceptor moiety comprising a GlcNAcβ1-3-Galβ1-4-Glcresidue in the presence of a β1-4-galactosyltransferase; and

c) contacting a reaction mixture comprising an activated GalNAc (i.eUDP-GalNAc) to the acceptor moiety comprising aGalβ1-4-GlcNAcβ1-3-Galβ1-4-Glc residue in the presence of thepolyglycosyltransferase of step a). A suitable β1-4galactosyltransferase can be isolated from bovine milk.

Oligosaccharide synthesis using a polyglycosyltransferase is generallyconducted at a temperature of from 15 to 38° C., preferably from 20 to25° C. While enzymatic activity is comparable at 25° C. and 37° C., thepolyglycosyltransferase stability is greater at 25° C.

In a preferred embodiment the polyglycosyltransferase activity isobserved in the absence of α-lactalbumin.

In a preferred embodiment the polyglycosyltransferase activity isobserved at the same pH, more preferably at pH 6.5 to 7.5.

In a preferred embodiment polyglycosyltransferase activity is observedat the same temperature.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLE 1

Synthesis of GalNAcβ1-3-Galβ1-4-GlcNAcβ1-3-Galβ1-4-Glc

Lactose was contacted with UDP-N-acetylglucosamine and a β-galactosideβ1-3 N-acetylglucosaminyl transferase of SEQ ID NO: 3, in a 0.5 M HEPESbuffered aqueous solution at 25° C. The product trisaccharide was thencontacted with UDP-Gal and a β-N-acetylglucosaminoside β1-4Galactosyltransferase isolated from bovine milk, in a 0.05 M HEPESbuffered aqueous solution at 37° C. The product tetrasaccharide was thencontacted with UDP-N-acetylgalactosamine and a β-galactoside β1-3N-acetylgalactosaminyl transferase of SEQ ID NO: 3, in a 0.05 M HEPESbuffered aqueous solution at 25° C. The title pentasaccharide wasisolated by conventional methods.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedtherein.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 8                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5859 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE:                                                   - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (B) STRAIN: F62                                                      - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..381                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 445..1491                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 2342..3262                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 3322..4335                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 4354..5196                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CTG CAG GCC GTC GCC GTA TTC AAA CAA CTG CC - #C GAA GCC GCC GCG CTC           48                                                                       Leu Gln Ala Val Ala Val Phe Lys Gln Leu Pr - #o Glu Ala Ala Ala Leu             1               5 - #                 10 - #                 15              - - GCC GCC GCC AAC AAA CGC GTG CAA AAC CTG CT - #G AAA AAA GCC GAT GCC           96                                                                       Ala Ala Ala Asn Lys Arg Val Gln Asn Leu Le - #u Lys Lys Ala Asp Ala                        20     - #             25     - #             30                  - - GCG TTG GGC GAA GTC AAT GAA AGC CTG CTG CA - #A CAG GAC GAA GAA AAA          144                                                                       Ala Leu Gly Glu Val Asn Glu Ser Leu Leu Gl - #n Gln Asp Glu Glu Lys                    35         - #         40         - #         45                      - - GCC CTG TAC GCT GCC GCG CAA GGT TTG CAG CC - #G AAA ATT GCC GCC GCC          192                                                                       Ala Leu Tyr Ala Ala Ala Gln Gly Leu Gln Pr - #o Lys Ile Ala Ala Ala               50              - #    55              - #    60                           - - GTC GCC GAA GGC AAT TTC CGA ACC GCC TTG TC - #C GAA CTG GCT TCC GTC          240                                                                       Val Ala Glu Gly Asn Phe Arg Thr Ala Leu Se - #r Glu Leu Ala Ser Val           65                  - #70                  - #75                  - #80        - - AAG CCG CAG GTT GAT GCC TTC TTC GAC GGC GT - #G ATG GTG ATG GCG GAA          288                                                                       Lys Pro Gln Val Asp Ala Phe Phe Asp Gly Va - #l Met Val Met Ala Glu                            85 - #                 90 - #                 95              - - GAT GCC GCC GTA AAA CAA AAC CGC CTG AAC CT - #G CTG AAC CGC TTG GCA          336                                                                       Asp Ala Ala Val Lys Gln Asn Arg Leu Asn Le - #u Leu Asn Arg Leu Ala                       100      - #           105      - #           110                  - - GAG CAG ATG AAC GCG GTG GCC GAC ATC GCG CT - #T TTG GGC GAG TAA              38 - #1                                                                   Glu Gln Met Asn Ala Val Ala Asp Ile Ala Le - #u Leu Gly Glu                           115          - #       120          - #       125                      - - CCGTTGTACA GTCCAAATGC CGTCTGAAGC CTTCAGGCGG CATCAAATTA TC -             #GGGAGAGT    441                                                                 - - AAA TTG CAG CCT TTA GTC AGC GTA TTG ATT TG - #C GCC TAC AAC GTA        GAA      489                                                                        Leu Gln Pro Leu Val Ser Val Leu - #Ile Cys Ala Tyr Asn Val Glu                 1            - #   5               - #   10               - #   15       - - AAA TAT TTT GCC CAA TCA TTA GCC GCC GTC GT - #G AAT CAG ACT TGG CGC          537                                                                       Lys Tyr Phe Ala Gln Ser Leu Ala Ala Val Va - #l Asn Gln Thr Trp Arg                            20 - #                 25 - #                 30              - - AAC TTG GAT ATT TTG ATT GTC GAT GAC GGC TC - #G ACA GAC GGC ACA CTT          585                                                                       Asn Leu Asp Ile Leu Ile Val Asp Asp Gly Se - #r Thr Asp Gly Thr Leu                        35     - #             40     - #             45                  - - GCC ATT GCC AAG GAT TTT CAA AAG CGG GAC AG - #C CGT ATC AAA ATC CTT          633                                                                       Ala Ile Ala Lys Asp Phe Gln Lys Arg Asp Se - #r Arg Ile Lys Ile Leu                    50         - #         55         - #         60                      - - GCA CAA GCT CAA AAT TCC GGC CTG ATT CCC TC - #T TTA AAC ATC GGG CTG          681                                                                       Ala Gln Ala Gln Asn Ser Gly Leu Ile Pro Se - #r Leu Asn Ile Gly Leu                65             - #     70             - #     75                          - - GAC GAA TTG GCA AAG TCG GGG GGG GGG GGG GG - #G GAA TAT ATT GCG CGC          729                                                                       Asp Glu Leu Ala Lys Ser Gly Gly Gly Gly Gl - #y Glu Tyr Ile Ala Arg            80                 - # 85                 - # 90                 - # 95       - - ACC GAT GCC GAC GAT ATT GCC TCC CCC GGC TG - #G ATT GAG AAA ATC GTG          777                                                                       Thr Asp Ala Asp Asp Ile Ala Ser Pro Gly Tr - #p Ile Glu Lys Ile Val                           100  - #               105  - #               110              - - GGC GAG ATG GAA AAA GAC CGC AGC ATC ATT GC - #G ATG GGC GCG TGG CTG          825                                                                       Gly Glu Met Glu Lys Asp Arg Ser Ile Ile Al - #a Met Gly Ala Trp Leu                       115      - #           120      - #           125                  - - GAA GTT TTG TCG GAA GAA AAG GAC GGC AAC CG - #G CTG GCG CGG CAC CAC          873                                                                       Glu Val Leu Ser Glu Glu Lys Asp Gly Asn Ar - #g Leu Ala Arg His His                   130          - #       135          - #       140                      - - AAA CAC GGC AAA ATT TGG AAA AAG CCG ACC CG - #G CAC GAA GAC ATC GCC          921                                                                       Lys His Gly Lys Ile Trp Lys Lys Pro Thr Ar - #g His Glu Asp Ile Ala               145              - #   150              - #   155                          - - GCC TTT TTC CCT TTC GGC AAC CCC ATA CAC AA - #C AAC ACG ATG ATT ATG          969                                                                       Ala Phe Phe Pro Phe Gly Asn Pro Ile His As - #n Asn Thr Met Ile Met           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - CGG CGC AGC GTC ATT GAC GGC GGT TTG CGT TA - #C GAC ACC GAG CGG        GAT     1017                                                                    Arg Arg Ser Val Ile Asp Gly Gly Leu Arg Ty - #r Asp Thr Glu Arg Asp                          180  - #               185  - #               190              - - TGG GCG GAA GAT TAC CAA TTT TGG TAC GAT GT - #C AGC AAA TTG GGC AGG         1065                                                                       Trp Ala Glu Asp Tyr Gln Phe Trp Tyr Asp Va - #l Ser Lys Leu Gly Arg                       195      - #           200      - #           205                  - - CTG GCT TAT TAT CCC GAA GCC TTG GTC AAA TA - #C CGC CTT CAC GCC AAT         1113                                                                       Leu Ala Tyr Tyr Pro Glu Ala Leu Val Lys Ty - #r Arg Leu His Ala Asn                   210          - #       215          - #       220                      - - CAG GTT TCA TCC AAA CAC AGC GTC CGC CAA CA - #C GAA ATC GCG CAA GGC         1161                                                                       Gln Val Ser Ser Lys His Ser Val Arg Gln Hi - #s Glu Ile Ala Gln Gly               225              - #   230              - #   235                          - - ATC CAA AAA ACC GCC AGA AAC GAT TTT TTG CA - #G TCT ATG GGT TTT AAA         1209                                                                       Ile Gln Lys Thr Ala Arg Asn Asp Phe Leu Gl - #n Ser Met Gly Phe Lys           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - ACC CGG TTC GAC AGC CTA GAA TAC CGC CAA AC - #A AAA GCA GCG GCG        TAT     1257                                                                    Thr Arg Phe Asp Ser Leu Glu Tyr Arg Gln Th - #r Lys Ala Ala Ala Tyr                          260  - #               265  - #               270              - - GAA CTG CCG GAG AAG GAT TTG CCG GAA GAA GA - #T TTT GAA CGC GCC CGC         1305                                                                       Glu Leu Pro Glu Lys Asp Leu Pro Glu Glu As - #p Phe Glu Arg Ala Arg                       275      - #           280      - #           285                  - - CGG TTT TTG TAC CAA TGC TTC AAA CGG ACG GA - #C ACG CCG CCC TCC GGC         1353                                                                       Arg Phe Leu Tyr Gln Cys Phe Lys Arg Thr As - #p Thr Pro Pro Ser Gly                   290          - #       295          - #       300                      - - GCG TGG CTG GAT TTC GCG GCA GAC GGC AGG AT - #G AGG CGG CTG TTT ACC         1401                                                                       Ala Trp Leu Asp Phe Ala Ala Asp Gly Arg Me - #t Arg Arg Leu Phe Thr               305              - #   310              - #   315                          - - TTG AGG CAA TAC TTC GGC ATT TTG TAC CGG CT - #G ATT AAA AAC CGC CGG         1449                                                                       Leu Arg Gln Tyr Phe Gly Ile Leu Tyr Arg Le - #u Ile Lys Asn Arg Arg           320                 3 - #25                 3 - #30                 3 -      #35                                                                              - - CAG GCG CGG TCG GAT TCG GCA GGG AAA GAA CA - #G GAG ATT TAA                 - #1491                                                                   Gln Ala Arg Ser Asp Ser Ala Gly Lys Glu Gl - #n Glu Ile                                       340  - #               345                                     - - TGCAAAACCA CGTTATCAGC TTGGCTTCCG CCGCAGAACG CAGGGCGCAC AT -             #TGCCGCAA   1551                                                                 - - CCTTCGGCAG TCGCGGCATC CCGTTCCAGT TTTTCGACGC ACTGATGCCG TC -            #TGAAAGGC   1611                                                                 - - TGGAACGGGC AATGGCGGAA CTCGTCCCCG GCTTGTCGGC GCACCCCTAT TT -            #GAGCGGAG   1671                                                                 - - TGGAAAAAGC CTGCTTTATG AGCCACGCCG TATTGTGGGA ACAGGCATTG GA -            #CGAAGGCG   1731                                                                 - - TACCGTATAT CGCCGTATTT GAAGATGATG TCTTACTCGG CGAAGGCGCG GA -            #GCAGTTCC   1791                                                                 - - TTGCCGAAGA TACTTGGCTG CAAGAACGCT TTGACCCCGA TTCCGCCTTT GT -            #CGTCCGCT   1851                                                                 - - TGGAAACGAT GTTTATGCAC GTCCTGACCT CGCCCTCCGG CGTGGCGGAC TA -            #CGGCGGGC   1911                                                                 - - GCGCCTTTCC GCTTTTGGAA AGCGAACACT GCGGGACGGC GGGCTATATT AT -            #TTCCCGAA   1971                                                                 - - AGGCGATGCG TTTTTTCTTG GACAGGTTTG CCGTTTTGCC GCCCGAACGC CT -            #GCACCCTG   2031                                                                 - - TCGATTTGAT GATGTTCGGC AACCCTGACG ACAGGGAAGG AATGCCGGTT TG -            #CCAGCTCA   2091                                                                 - - ATCCCGCCTT GTGCGCCCAA GAGCTGCATT ATGCCAAGTT TCACGACCAA AA -            #CAGCGCAT   2151                                                                 - - TGGGCAGCCT GATCGAACAT GACCGCCGCC TGAACCGCAA ACAGCAATGG CG -            #CGATTCCC   2211                                                                 - - CCGCCAACAC ATTCAAACAC CGCCTGATCC GCGCCTTGAC CAAAATCGGC AG -            #GGAAAGGG   2271                                                                 - - AAAAACGCCG GCAAAGGCGC GAACAGTTAA TCGGCAAGAT TATTGTGCCT TT -            #CCAATAAA   2331                                                                 - - AGGAGAAAAG ATG GAC ATC GTA TTT GCG GCA GAC GA - #C AAC TAT GCC GCC          2380                                                                       - -            Met Asp Ile Val - #Phe Ala Ala Asp Asp Asn Tyr Ala Ala                     1     - #          5        - #          10                       - - TAC CTT TGC GTT GCG GCA AAA AGC GTG GAA GC - #G GCC CAT CCC GAT ACG         2428                                                                       Tyr Leu Cys Val Ala Ala Lys Ser Val Glu Al - #a Ala His Pro Asp Thr                15             - #     20             - #     25                          - - GAA ATC AGG TTC CAC GTC CTC GAT GCC GGC AT - #C AGT GAG GAA AAC CGG         2476                                                                       Glu Ile Arg Phe His Val Leu Asp Ala Gly Il - #e Ser Glu Glu Asn Arg            30                 - # 35                 - # 40                 - # 45       - - GCG GCG GTT GCC GCC AAT TTG CGG GGG GGG GG - #T AAT ATC CGC TTT ATA         2524                                                                       Ala Ala Val Ala Ala Asn Leu Arg Gly Gly Gl - #y Asn Ile Arg Phe Ile                            50 - #                 55 - #                 60              - - GAC GTA AAC CCC GAA GAT TTC GCC GGC TTC CC - #C TTA AAC ATC AGG CAC         2572                                                                       Asp Val Asn Pro Glu Asp Phe Ala Gly Phe Pr - #o Leu Asn Ile Arg His                        65     - #             70     - #             75                  - - ATT TCC ATT ACG ACT TAT GCC CGC CTG AAA TT - #G GGC GAA TAC ATT GCC         2620                                                                       Ile Ser Ile Thr Thr Tyr Ala Arg Leu Lys Le - #u Gly Glu Tyr Ile Ala                    80         - #         85         - #         90                      - - GAT TGC GAC AAA GTC CTG TAT CTG GAT ACG GA - #C GTA TTG GTC AGG GAC         2668                                                                       Asp Cys Asp Lys Val Leu Tyr Leu Asp Thr As - #p Val Leu Val Arg Asp                95             - #    100             - #    105                          - - GGC CTG AAG CCC TTA TGG GAT ACC GAT TTG GG - #C GGT AAC TGG GTC GGC         2716                                                                       Gly Leu Lys Pro Leu Trp Asp Thr Asp Leu Gl - #y Gly Asn Trp Val Gly           110                 1 - #15                 1 - #20                 1 -      #25                                                                              - - GCG TGC ATC GAT TTG TTT GTC GAA AGG CAG GA - #A GGA TAC AAA CAA        AAA     2764                                                                    Ala Cys Ile Asp Leu Phe Val Glu Arg Gln Gl - #u Gly Tyr Lys Gln Lys                          130  - #               135  - #               140              - - ATC GGT ATG GCG GAC GGA GAA TAT TAT TTC AA - #T GCC GGC GTA TTG CTG         2812                                                                       Ile Gly Met Ala Asp Gly Glu Tyr Tyr Phe As - #n Ala Gly Val Leu Leu                       145      - #           150      - #           155                  - - ATC AAC CTG AAA AAG TGG CGG CGG CAC GAT AT - #T TTC AAA ATG TCC TGC         2860                                                                       Ile Asn Leu Lys Lys Trp Arg Arg His Asp Il - #e Phe Lys Met Ser Cys                   160          - #       165          - #       170                      - - GAA TGG GTG GAA CAA TAC AAG GAC GTG ATG CA - #A TAT CAG GAT CAG GAC         2908                                                                       Glu Trp Val Glu Gln Tyr Lys Asp Val Met Gl - #n Tyr Gln Asp Gln Asp               175              - #   180              - #   185                          - - ATT TTG AAC GGG CTG TTT AAA GGC GGG GTG TG - #T TAT GCG AAC AGC CGT         2956                                                                       Ile Leu Asn Gly Leu Phe Lys Gly Gly Val Cy - #s Tyr Ala Asn Ser Arg           190                 1 - #95                 2 - #00                 2 -      #05                                                                              - - TTC AAC TTT ATG CCG ACC AAT TAT GCC TTT AT - #G GCG AAC GGG TTT        GCG     3004                                                                    Phe Asn Phe Met Pro Thr Asn Tyr Ala Phe Me - #t Ala Asn Gly Phe Ala                          210  - #               215  - #               220              - - TCC CGC CAT ACC GAC CCG CTT TAC CTC GAC CG - #T ACC AAT ACG GCG ATG         3052                                                                       Ser Arg His Thr Asp Pro Leu Tyr Leu Asp Ar - #g Thr Asn Thr Ala Met                       225      - #           230      - #           235                  - - CCC GTC GCC GTC AGC CAT TAT TGC GGC TCG GC - #A AAG CCG TGG CAC AGG         3100                                                                       Pro Val Ala Val Ser His Tyr Cys Gly Ser Al - #a Lys Pro Trp His Arg                   240          - #       245          - #       250                      - - GAC TGC ACC GTT TGG GGT GCG GAA CGT TTC AC - #A GAG TTG GCC GGC AGC         3148                                                                       Asp Cys Thr Val Trp Gly Ala Glu Arg Phe Th - #r Glu Leu Ala Gly Ser               255              - #   260              - #   265                          - - CTG ACG ACC GTT CCC GAA GAA TGG CGC GGC AA - #A CTT GCC GTC CCG CCG         3196                                                                       Leu Thr Thr Val Pro Glu Glu Trp Arg Gly Ly - #s Leu Ala Val Pro Pro           270                 2 - #75                 2 - #80                 2 -      #85                                                                              - - ACA AAG TGT ATG CTT CAA AGA TGG CGC AAA AA - #G CTG TCT GCC AGA        TTC     3244                                                                    Thr Lys Cys Met Leu Gln Arg Trp Arg Lys Ly - #s Leu Ser Ala Arg Phe                          290  - #               295  - #               300              - - TTA CGC AAG ATT TAT TGA CGGGGCAGGC CGTCTGAAGC CT - #TCAGACGG                3292                                                                       Leu Arg Lys Ile Tyr                                                                       305                                                                - - CATCGGACGT ATCGGAAAGG AGAAACGGA TTG CAG CCT TTA GTC - # AGC GTA TTG         3345                                                                                         - #              Leu Gln P - #ro Leu Val Ser Val Leu                          - #                1  - #             5                      - - ATT TGC GCC TAC AAC GCA GAA AAA TAT TTT GC - #C CAA TCA TTG GCC GCC         3393                                                                       Ile Cys Ala Tyr Asn Ala Glu Lys Tyr Phe Al - #a Gln Ser Leu Ala Ala                10             - #     15             - #     20                          - - GTA GTG GGG CAG ACT TGG CGC AAC TTG GAT AT - #T TTG ATT GTC GAT GAC         3441                                                                       Val Val Gly Gln Thr Trp Arg Asn Leu Asp Il - #e Leu Ile Val Asp Asp            25                 - # 30                 - # 35                 - # 40       - - GGC TCG ACG GAC GGC ACG CCC GCC ATT GCC CG - #G CAT TTC CAA GAA CAG         3489                                                                       Gly Ser Thr Asp Gly Thr Pro Ala Ile Ala Ar - #g His Phe Gln Glu Gln                            45 - #                 50 - #                 55              - - GAC GGC AGG ATC AGG ATA ATT TCC AAT CCC CG - #C AAT TTG GGC TTT ATC         3537                                                                       Asp Gly Arg Ile Arg Ile Ile Ser Asn Pro Ar - #g Asn Leu Gly Phe Ile                        60     - #             65     - #             70                  - - GCC TCT TTA AAC ATC GGG CTG GAC GAA TTG GC - #A AAG TCG GGG GGG GGG         3585                                                                       Ala Ser Leu Asn Ile Gly Leu Asp Glu Leu Al - #a Lys Ser Gly Gly Gly                    75         - #         80         - #         85                      - - GAA TAT ATT GCG CGC ACC GAT GCC GAC GAT AT - #T GCC TCC CCC GGC TGG         3633                                                                       Glu Tyr Ile Ala Arg Thr Asp Ala Asp Asp Il - #e Ala Ser Pro Gly Trp                90             - #     95             - #    100                          - - ATT GAG AAA ATC GTG GGC GAG ATG GAA AAA GA - #C CGC AGC ATC ATT GCG         3681                                                                       Ile Glu Lys Ile Val Gly Glu Met Glu Lys As - #p Arg Ser Ile Ile Ala           105                 1 - #10                 1 - #15                 1 -      #20                                                                              - - ATG GGC GCG TGG TTG GAA GTT TTG TCG GAA GA - #A AAC AAT AAA AGC        GTG     3729                                                                    Met Gly Ala Trp Leu Glu Val Leu Ser Glu Gl - #u Asn Asn Lys Ser Val                          125  - #               130  - #               135              - - CTT GCC GCC ATT GCC CGA AAC GGC GCA ATT TG - #G GAC AAA CCG ACC CGG         3777                                                                       Leu Ala Ala Ile Ala Arg Asn Gly Ala Ile Tr - #p Asp Lys Pro Thr Arg                       140      - #           145      - #           150                  - - CAT GAA GAC ATT GTC GCC GTT TTC CCT TTC GG - #C AAC CCC ATA CAC AAC         3825                                                                       His Glu Asp Ile Val Ala Val Phe Pro Phe Gl - #y Asn Pro Ile His Asn                   155          - #       160          - #       165                      - - AAC ACG ATG ATT ATG AGG CGC AGC GTC ATT GA - #C GGC GGT TTG CGG TTC         3873                                                                       Asn Thr Met Ile Met Arg Arg Ser Val Ile As - #p Gly Gly Leu Arg Phe               170              - #   175              - #   180                          - - GAT CCA GCC TAT ATC CAC GCC GAA GAC TAT AA - #G TTT TGG TAC GAA GCC         3921                                                                       Asp Pro Ala Tyr Ile His Ala Glu Asp Tyr Ly - #s Phe Trp Tyr Glu Ala           185                 1 - #90                 1 - #95                 2 -      #00                                                                              - - GGC AAA CTG GGC AGG CTG GCT TAT TAT CCC GA - #A GCC TTG GTC AAA        TAC     3969                                                                    Gly Lys Leu Gly Arg Leu Ala Tyr Tyr Pro Gl - #u Ala Leu Val Lys Tyr                          205  - #               210  - #               215              - - CGC TTC CAT CAA GAC CAG ACT TCT TCC AAA TA - #C AAC CTG CAA CAG CGC         4017                                                                       Arg Phe His Gln Asp Gln Thr Ser Ser Lys Ty - #r Asn Leu Gln Gln Arg                       220      - #           225      - #           230                  - - AGG ACG GCG TGG AAA ATC AAA GAA GAA ATC AG - #G GCG GGG TAT TGG AAG         4065                                                                       Arg Thr Ala Trp Lys Ile Lys Glu Glu Ile Ar - #g Ala Gly Tyr Trp Lys                   235          - #       240          - #       245                      - - GCG GCA GGC ATA GCC GTC GGG GCG GAC TGC CT - #G AAT TAC GGG CTT TTG         4113                                                                       Ala Ala Gly Ile Ala Val Gly Ala Asp Cys Le - #u Asn Tyr Gly Leu Leu               250              - #   255              - #   260                          - - AAA TCA ACG GCA TAT GCG TTG TAC GAA AAA GC - #C TTG TCC GGA CAG GAT         4161                                                                       Lys Ser Thr Ala Tyr Ala Leu Tyr Glu Lys Al - #a Leu Ser Gly Gln Asp           265                 2 - #70                 2 - #75                 2 -      #80                                                                              - - ATC GGA TGC CTC CGC CTG TTC CTG TAC GAA TA - #T TTC TTG TCG TTG        GAA     4209                                                                    Ile Gly Cys Leu Arg Leu Phe Leu Tyr Glu Ty - #r Phe Leu Ser Leu Glu                          285  - #               290  - #               295              - - AAG TAT TCT TTG ACC GAT TTG CTG GAT TTC TT - #G ACA GAC CGC GTG ATG         4257                                                                       Lys Tyr Ser Leu Thr Asp Leu Leu Asp Phe Le - #u Thr Asp Arg Val Met                       300      - #           305      - #           310                  - - AGG AAG CTG TTT GCC GCA CCG CAA TAT AGG AA - #A ATC CTG AAA AAA ATG         4305                                                                       Arg Lys Leu Phe Ala Ala Pro Gln Tyr Arg Ly - #s Ile Leu Lys Lys Met                   315          - #       320          - #       325                      - - TTA CGC CCT TGG AAA TAC CGC AGC TAT TGA AA - #CCGAACAG GATAAATC ATG         4356                                                                       Leu Arg Pro Trp Lys Tyr Arg Ser Tyr    - #                  - #  Met              330              - #   335              - #                  - #   1       - - CAA AAC CAC GTT ATC AGC TTG GCT TCC GCC GC - #A GAG CGC AGG GCG CAC         4404                                                                       Gln Asn His Val Ile Ser Leu Ala Ser Ala Al - #a Glu Arg Arg Ala His                         5    - #              10    - #              15                  - - ATT GCC GAT ACC TTC GGC AGT CGC GGC ATC CC - #G TTC CAG TTT TTC GAC         4452                                                                       Ile Ala Asp Thr Phe Gly Ser Arg Gly Ile Pr - #o Phe Gln Phe Phe Asp                    20         - #         25         - #         30                      - - GCA CTG ATG CCG TCT GAA AGG CTG GAA CAG GC - #G ATG GCG GAA CTC GTC         4500                                                                       Ala Leu Met Pro Ser Glu Arg Leu Glu Gln Al - #a Met Ala Glu Leu Val                35             - #     40             - #     45                          - - CCC GGC TTG TCG GCG CAC CCC TAT TTG AGC GG - #A GTG GAA AAA GCC TGC         4548                                                                       Pro Gly Leu Ser Ala His Pro Tyr Leu Ser Gl - #y Val Glu Lys Ala Cys            50                 - # 55                 - # 60                 - # 65       - - TTT ATG AGC CAC GCC GTA TTG TGG GAA CAG GC - #G TTG GAT GAA GGT CTG         4596                                                                       Phe Met Ser His Ala Val Leu Trp Glu Gln Al - #a Leu Asp Glu Gly Leu                            70 - #                 75 - #                 80              - - CCG TAT ATC GCC GTA TTT GAG GAC GAC GTT TT - #A CTC GGC GAA GGC GCG         4644                                                                       Pro Tyr Ile Ala Val Phe Glu Asp Asp Val Le - #u Leu Gly Glu Gly Ala                        85     - #             90     - #             95                  - - GAG CAG TTC CTT GCC GAA GAT ACT TGG TTG GA - #A GAG CGT TTT GAC AAG         4692                                                                       Glu Gln Phe Leu Ala Glu Asp Thr Trp Leu Gl - #u Glu Arg Phe Asp Lys                   100          - #       105          - #       110                      - - GAT TCC GCC TTT ATC GTC CGT TTG GAA ACG AT - #G TTT GCG AAA GTT ATT         4740                                                                       Asp Ser Ala Phe Ile Val Arg Leu Glu Thr Me - #t Phe Ala Lys Val Ile               115              - #   120              - #   125                          - - GTC AGA CCG GAT AAA GTC CTG AAT TAT GAA AA - #C CGG TCA TTT CCT TTG         4788                                                                       Val Arg Pro Asp Lys Val Leu Asn Tyr Glu As - #n Arg Ser Phe Pro Leu           130                 1 - #35                 1 - #40                 1 -      #45                                                                              - - CTG GAG AGC GAA CAT TGT GGG ACG GCT GGC TA - #T ATC ATT TCG CGT        GAG     4836                                                                    Leu Glu Ser Glu His Cys Gly Thr Ala Gly Ty - #r Ile Ile Ser Arg Glu                          150  - #               155  - #               160              - - GCG ATG CGG TTT TTC TTG GAC AGG TTT GCC GT - #T TTG CCG CCA GAG CGG         4884                                                                       Ala Met Arg Phe Phe Leu Asp Arg Phe Ala Va - #l Leu Pro Pro Glu Arg                       165      - #           170      - #           175                  - - ATT AAA GCG GTA GAT TTG ATG ATG TTT ACT TA - #T TTC TTT GAT AAG GAG         4932                                                                       Ile Lys Ala Val Asp Leu Met Met Phe Thr Ty - #r Phe Phe Asp Lys Glu                   180          - #       185          - #       190                      - - GGG ATG CCT GTT TAT CAG GTT AGT CCC GCC TT - #A TGT ACC CAA GAA TTG         4980                                                                       Gly Met Pro Val Tyr Gln Val Ser Pro Ala Le - #u Cys Thr Gln Glu Leu               195              - #   200              - #   205                          - - CAT TAT GCC AAG TTT CTC AGT CAA AAC AGT AT - #G TTG GGT AGC GAT TTG         5028                                                                       His Tyr Ala Lys Phe Leu Ser Gln Asn Ser Me - #t Leu Gly Ser Asp Leu           210                 2 - #15                 2 - #20                 2 -      #25                                                                              - - GAA AAA GAT AGG GAA CAA GGA AGA AGA CAC CG - #C CGT TCG TTG AAG        GTG     5076                                                                    Glu Lys Asp Arg Glu Gln Gly Arg Arg His Ar - #g Arg Ser Leu Lys Val                          230  - #               235  - #               240              - - ATG TTT GAC TTG AAG CGT GCT TTG GGT AAA TT - #C GGT AGG GAA AAG AAG         5124                                                                       Met Phe Asp Leu Lys Arg Ala Leu Gly Lys Ph - #e Gly Arg Glu Lys Lys                       245      - #           250      - #           255                  - - AAA AGA ATG GAG CGT CAA AGG CAG GCG GAG CT - #T GAG AAA GTT TAC GGC         5172                                                                       Lys Arg Met Glu Arg Gln Arg Gln Ala Glu Le - #u Glu Lys Val Tyr Gly                   260          - #       265          - #       270                      - - AGG CGG GTC ATA TTG TTC AAA TAG TTTGTGTAAA AT - #ATAGGGGA TTAAAATCAG        5226                                                                       Arg Arg Val Ile Leu Phe Lys                                                       275              - #   280                                                 - - AAATGGACAC ACTGTCATTC CCGCGCAGGC GGGAATCTAG GTCTTTAAAC TT -             #CGGTTTTT   5286                                                                 - - TCCGATAAAT TCTTGCCGCA TTAAAATTCC AGATTCCCGC TTTCGCGGGG AT -            #GACGGCGG   5346                                                                 - - GGGGATTGTT GCTTTTTCGG ATAAAATCCC GTGTTTTTTC ATCTGCTAGG TA -            #AAATCGCC   5406                                                                 - - CCAAAGCGTC TGCATCGCGG CGATGGCGGC GAGTGGGGCG GTTTCTGTGC GT -            #AAAATCCG   5466                                                                 - - TTTTCCGAGT GTAACCGCCT GAAAGCCGGC TTCAAATGCC TGTTGTTCTT CC -            #TGTTCTGT   5526                                                                 - - CCAGCCGCCT TCGGGCCCGA CCATAAAGAC GATTGCGCCG GACGGGTGGC GG -            #ATGTCGCC   5586                                                                 - - GAGTTTGCAG GCGCGGTTGA TGCTCATAAT CAGCTTGGTG TTTTCAGACG GC -            #ATTTTGTC   5646                                                                 - - GAGTGCTTCA CGGTAGCCGA TGATGGGCAG TACGGGGGGA ACGGTGTTCC TG -            #CCGCTTTG   5706                                                                 - - TTCGCACGCG GAGATGACGA TTTCCTGCCA GCGTGCGAGG CGTTTGGCGG CG -            #CGTTCTCC   5766                                                                 - - GTCGAGGCGG ACGATGCAGC GTTCGCTGAT GACGGGCTGT ATGGCGGTTA CG -            #CCGAGTTC   5826                                                                 - - GACGCTTTTT TGCAGGGTGA AATCCATGCG ATC       - #                  -      #       5859                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 126 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Leu Gln Ala Val Ala Val Phe Lys Gln Leu Pr - #o Glu Ala Ala Ala        Leu                                                                               1               5 - #                 10 - #                 15             - - Ala Ala Ala Asn Lys Arg Val Gln Asn Leu Le - #u Lys Lys Ala Asp Ala                   20     - #             25     - #             30                  - - Ala Leu Gly Glu Val Asn Glu Ser Leu Leu Gl - #n Gln Asp Glu Glu Lys               35         - #         40         - #         45                      - - Ala Leu Tyr Ala Ala Ala Gln Gly Leu Gln Pr - #o Lys Ile Ala Ala Ala           50             - #     55             - #     60                          - - Val Ala Glu Gly Asn Phe Arg Thr Ala Leu Se - #r Glu Leu Ala Ser Val       65                 - # 70                 - # 75                 - # 80       - - Lys Pro Gln Val Asp Ala Phe Phe Asp Gly Va - #l Met Val Met Ala Glu                       85 - #                 90 - #                 95              - - Asp Ala Ala Val Lys Gln Asn Arg Leu Asn Le - #u Leu Asn Arg Leu Ala                  100      - #           105      - #           110                  - - Glu Gln Met Asn Ala Val Ala Asp Ile Ala Le - #u Leu Gly Glu                      115          - #       120          - #       125                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 348 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Leu Gln Pro Leu Val Ser Val Leu Ile Cys Al - #a Tyr Asn Val Glu Lys        1               5 - #                 10 - #                 15              - - Tyr Phe Ala Gln Ser Leu Ala Ala Val Val As - #n Gln Thr Trp Arg Asn                   20     - #             25     - #             30                  - - Leu Asp Ile Leu Ile Val Asp Asp Gly Ser Th - #r Asp Gly Thr Leu Ala               35         - #         40         - #         45                      - - Ile Ala Lys Asp Phe Gln Lys Arg Asp Ser Ar - #g Ile Lys Ile Leu Ala           50             - #     55             - #     60                          - - Gln Ala Gln Asn Ser Gly Leu Ile Pro Ser Le - #u Asn Ile Gly Leu Asp       65                 - # 70                 - # 75                 - # 80       - - Glu Leu Ala Lys Ser Gly Gly Gly Gly Gly Gl - #u Tyr Ile Ala Arg Thr                       85 - #                 90 - #                 95              - - Asp Ala Asp Asp Ile Ala Ser Pro Gly Trp Il - #e Glu Lys Ile Val Gly                  100      - #           105      - #           110                  - - Glu Met Glu Lys Asp Arg Ser Ile Ile Ala Me - #t Gly Ala Trp Leu Glu              115          - #       120          - #       125                      - - Val Leu Ser Glu Glu Lys Asp Gly Asn Arg Le - #u Ala Arg His His Lys          130              - #   135              - #   140                          - - His Gly Lys Ile Trp Lys Lys Pro Thr Arg Hi - #s Glu Asp Ile Ala Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Phe Phe Pro Phe Gly Asn Pro Ile His Asn As - #n Thr Met Ile Met        Arg                                                                                             165  - #               170  - #               175             - - Arg Ser Val Ile Asp Gly Gly Leu Arg Tyr As - #p Thr Glu Arg Asp Trp                  180      - #           185      - #           190                  - - Ala Glu Asp Tyr Gln Phe Trp Tyr Asp Val Se - #r Lys Leu Gly Arg Leu              195          - #       200          - #       205                      - - Ala Tyr Tyr Pro Glu Ala Leu Val Lys Tyr Ar - #g Leu His Ala Asn Gln          210              - #   215              - #   220                          - - Val Ser Ser Lys His Ser Val Arg Gln His Gl - #u Ile Ala Gln Gly Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gln Lys Thr Ala Arg Asn Asp Phe Leu Gln Se - #r Met Gly Phe Lys        Thr                                                                                             245  - #               250  - #               255             - - Arg Phe Asp Ser Leu Glu Tyr Arg Gln Thr Ly - #s Ala Ala Ala Tyr Glu                  260      - #           265      - #           270                  - - Leu Pro Glu Lys Asp Leu Pro Glu Glu Asp Ph - #e Glu Arg Ala Arg Arg              275          - #       280          - #       285                      - - Phe Leu Tyr Gln Cys Phe Lys Arg Thr Asp Th - #r Pro Pro Ser Gly Ala          290              - #   295              - #   300                          - - Trp Leu Asp Phe Ala Ala Asp Gly Arg Met Ar - #g Arg Leu Phe Thr Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Arg Gln Tyr Phe Gly Ile Leu Tyr Arg Leu Il - #e Lys Asn Arg Arg        Gln                                                                                             325  - #               330  - #               335             - - Ala Arg Ser Asp Ser Ala Gly Lys Glu Gln Gl - #u Ile                                  340      - #           345                                         - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 306 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Asp Ile Val Phe Ala Ala Asp Asp Asn Ty - #r Ala Ala Tyr Leu Cys        1               5 - #                 10 - #                 15              - - Val Ala Ala Lys Ser Val Glu Ala Ala His Pr - #o Asp Thr Glu Ile Arg                   20     - #             25     - #             30                  - - Phe His Val Leu Asp Ala Gly Ile Ser Glu Gl - #u Asn Arg Ala Ala Val               35         - #         40         - #         45                      - - Ala Ala Asn Leu Arg Gly Gly Gly Asn Ile Ar - #g Phe Ile Asp Val Asn           50             - #     55             - #     60                          - - Pro Glu Asp Phe Ala Gly Phe Pro Leu Asn Il - #e Arg His Ile Ser Ile       65                 - # 70                 - # 75                 - # 80       - - Thr Thr Tyr Ala Arg Leu Lys Leu Gly Glu Ty - #r Ile Ala Asp Cys Asp                       85 - #                 90 - #                 95              - - Lys Val Leu Tyr Leu Asp Thr Asp Val Leu Va - #l Arg Asp Gly Leu Lys                  100      - #           105      - #           110                  - - Pro Leu Trp Asp Thr Asp Leu Gly Gly Asn Tr - #p Val Gly Ala Cys Ile              115          - #       120          - #       125                      - - Asp Leu Phe Val Glu Arg Gln Glu Gly Tyr Ly - #s Gln Lys Ile Gly Met          130              - #   135              - #   140                          - - Ala Asp Gly Glu Tyr Tyr Phe Asn Ala Gly Va - #l Leu Leu Ile Asn Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Lys Trp Arg Arg His Asp Ile Phe Lys Me - #t Ser Cys Glu Trp        Val                                                                                             165  - #               170  - #               175             - - Glu Gln Tyr Lys Asp Val Met Gln Tyr Gln As - #p Gln Asp Ile Leu Asn                  180      - #           185      - #           190                  - - Gly Leu Phe Lys Gly Gly Val Cys Tyr Ala As - #n Ser Arg Phe Asn Phe              195          - #       200          - #       205                      - - Met Pro Thr Asn Tyr Ala Phe Met Ala Asn Gl - #y Phe Ala Ser Arg His          210              - #   215              - #   220                          - - Thr Asp Pro Leu Tyr Leu Asp Arg Thr Asn Th - #r Ala Met Pro Val Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Val Ser His Tyr Cys Gly Ser Ala Lys Pro Tr - #p His Arg Asp Cys        Thr                                                                                             245  - #               250  - #               255             - - Val Trp Gly Ala Glu Arg Phe Thr Glu Leu Al - #a Gly Ser Leu Thr Thr                  260      - #           265      - #           270                  - - Val Pro Glu Glu Trp Arg Gly Lys Leu Ala Va - #l Pro Pro Thr Lys Cys              275          - #       280          - #       285                      - - Met Leu Gln Arg Trp Arg Lys Lys Leu Ser Al - #a Arg Phe Leu Arg Lys          290              - #   295              - #   300                          - - Ile Tyr                                                                  305                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 337 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Leu Gln Pro Leu Val Ser Val Leu Ile Cys Al - #a Tyr Asn Ala Glu Lys        1               5 - #                 10 - #                 15              - - Tyr Phe Ala Gln Ser Leu Ala Ala Val Val Gl - #y Gln Thr Trp Arg Asn                   20     - #             25     - #             30                  - - Leu Asp Ile Leu Ile Val Asp Asp Gly Ser Th - #r Asp Gly Thr Pro Ala               35         - #         40         - #         45                      - - Ile Ala Arg His Phe Gln Glu Gln Asp Gly Ar - #g Ile Arg Ile Ile Ser           50             - #     55             - #     60                          - - Asn Pro Arg Asn Leu Gly Phe Ile Ala Ser Le - #u Asn Ile Gly Leu Asp       65                 - # 70                 - # 75                 - # 80       - - Glu Leu Ala Lys Ser Gly Gly Gly Glu Tyr Il - #e Ala Arg Thr Asp Ala                       85 - #                 90 - #                 95              - - Asp Asp Ile Ala Ser Pro Gly Trp Ile Glu Ly - #s Ile Val Gly Glu Met                  100      - #           105      - #           110                  - - Glu Lys Asp Arg Ser Ile Ile Ala Met Gly Al - #a Trp Leu Glu Val Leu              115          - #       120          - #       125                      - - Ser Glu Glu Asn Asn Lys Ser Val Leu Ala Al - #a Ile Ala Arg Asn Gly          130              - #   135              - #   140                          - - Ala Ile Trp Asp Lys Pro Thr Arg His Glu As - #p Ile Val Ala Val Phe      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Phe Gly Asn Pro Ile His Asn Asn Thr Me - #t Ile Met Arg Arg        Ser                                                                                             165  - #               170  - #               175             - - Val Ile Asp Gly Gly Leu Arg Phe Asp Pro Al - #a Tyr Ile His Ala Glu                  180      - #           185      - #           190                  - - Asp Tyr Lys Phe Trp Tyr Glu Ala Gly Lys Le - #u Gly Arg Leu Ala Tyr              195          - #       200          - #       205                      - - Tyr Pro Glu Ala Leu Val Lys Tyr Arg Phe Hi - #s Gln Asp Gln Thr Ser          210              - #   215              - #   220                          - - Ser Lys Tyr Asn Leu Gln Gln Arg Arg Thr Al - #a Trp Lys Ile Lys Glu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Glu Ile Arg Ala Gly Tyr Trp Lys Ala Ala Gl - #y Ile Ala Val Gly        Ala                                                                                             245  - #               250  - #               255             - - Asp Cys Leu Asn Tyr Gly Leu Leu Lys Ser Th - #r Ala Tyr Ala Leu Tyr                  260      - #           265      - #           270                  - - Glu Lys Ala Leu Ser Gly Gln Asp Ile Gly Cy - #s Leu Arg Leu Phe Leu              275          - #       280          - #       285                      - - Tyr Glu Tyr Phe Leu Ser Leu Glu Lys Tyr Se - #r Leu Thr Asp Leu Leu          290              - #   295              - #   300                          - - Asp Phe Leu Thr Asp Arg Val Met Arg Lys Le - #u Phe Ala Ala Pro Gln      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Tyr Arg Lys Ile Leu Lys Lys Met Leu Arg Pr - #o Trp Lys Tyr Arg        Ser                                                                                             325  - #               330  - #               335             - - Tyr                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 280 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Gln Asn His Val Ile Ser Leu Ala Ser Al - #a Ala Glu Arg Arg Ala        1               5 - #                 10 - #                 15              - - His Ile Ala Asp Thr Phe Gly Ser Arg Gly Il - #e Pro Phe Gln Phe Phe                   20     - #             25     - #             30                  - - Asp Ala Leu Met Pro Ser Glu Arg Leu Glu Gl - #n Ala Met Ala Glu Leu               35         - #         40         - #         45                      - - Val Pro Gly Leu Ser Ala His Pro Tyr Leu Se - #r Gly Val Glu Lys Ala           50             - #     55             - #     60                          - - Cys Phe Met Ser His Ala Val Leu Trp Glu Gl - #n Ala Leu Asp Glu Gly       65                 - # 70                 - # 75                 - # 80       - - Leu Pro Tyr Ile Ala Val Phe Glu Asp Asp Va - #l Leu Leu Gly Glu Gly                       85 - #                 90 - #                 95              - - Ala Glu Gln Phe Leu Ala Glu Asp Thr Trp Le - #u Glu Glu Arg Phe Asp                  100      - #           105      - #           110                  - - Lys Asp Ser Ala Phe Ile Val Arg Leu Glu Th - #r Met Phe Ala Lys Val              115          - #       120          - #       125                      - - Ile Val Arg Pro Asp Lys Val Leu Asn Tyr Gl - #u Asn Arg Ser Phe Pro          130              - #   135              - #   140                          - - Leu Leu Glu Ser Glu His Cys Gly Thr Ala Gl - #y Tyr Ile Ile Ser Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Ala Met Arg Phe Phe Leu Asp Arg Phe Al - #a Val Leu Pro Pro        Glu                                                                                             165  - #               170  - #               175             - - Arg Ile Lys Ala Val Asp Leu Met Met Phe Th - #r Tyr Phe Phe Asp Lys                  180      - #           185      - #           190                  - - Glu Gly Met Pro Val Tyr Gln Val Ser Pro Al - #a Leu Cys Thr Gln Glu              195          - #       200          - #       205                      - - Leu His Tyr Ala Lys Phe Leu Ser Gln Asn Se - #r Met Leu Gly Ser Asp          210              - #   215              - #   220                          - - Leu Glu Lys Asp Arg Glu Gln Gly Arg Arg Hi - #s Arg Arg Ser Leu Lys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Val Met Phe Asp Leu Lys Arg Ala Leu Gly Ly - #s Phe Gly Arg Glu        Lys                                                                                             245  - #               250  - #               255             - - Lys Lys Arg Met Glu Arg Gln Arg Gln Ala Gl - #u Leu Glu Lys Val Tyr                  260      - #           265      - #           270                  - - Gly Arg Arg Val Ile Leu Phe Lys                                                  275          - #       280                                             - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Tyr Ser Arg Asp Ser Ser                                                    1               5                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 348 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Leu Gln Pro Leu Val Ser Val Leu Ile Cys Al - #a Tyr Asn Val Glu Lys      1               5   - #                10  - #                15               - - Tyr Phe Ala Gln Ser Leu Ala Ala Val Val As - #n Gln Thr Trp Arg Asn                  20      - #            25      - #            30                   - - Leu Asp Ile Leu Ile Val Asp Asp Gly Ser Th - #r Asp Gly Thr Leu Ala              35          - #        40          - #        45                       - - Ile Ala Lys Asp Phe Gln Lys Arg Asp Ser Ar - #g Ile Lys Ile Leu Ala          50              - #    55              - #    60                           - - Gln Ala Gln Asn Ser Gly Leu Ile Pro Ser Le - #u Asn Ile Gly Leu Asp      65                  - #70                  - #75                  - #80        - - Glu Leu Ala Lys Ser Gly Gly Gly Gly Gly Gl - #u Tyr Ile Ala Arg Thr                      85  - #                90  - #                95               - - Asp Ala Asp Asp Ile Ala Ser Pro Gly Trp Il - #e Glu Lys Ile Val Gly                  100      - #           105      - #           110                  - - Glu Met Glu Lys Asp Arg Ser Ile Ile Ala Me - #t Gly Ala Trp Leu Glu              115          - #       120          - #       125                      - - Val Leu Ser Glu Glu Lys Asp Gly Asn Arg Le - #u Ala Arg His His Lys          130              - #   135              - #   140                          - - His Gly Lys Ile Trp Lys Lys Pro Thr Arg Hi - #s Glu Asp Ile Ala Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Phe Phe Pro Phe Gly Asn Pro Ile His Asn As - #n Thr Met Ile Met        Arg                                                                                             165  - #               170  - #               175             - - Arg Ser Val Ile Asp Gly Gly Leu Arg Tyr As - #p Thr Glu Arg Asp Trp                  180      - #           185      - #           190                  - - Ala Glu Asp Tyr Gln Phe Trp Tyr Asp Val Se - #r Lys Leu Gly Arg Leu              195          - #       200          - #       205                      - - Ala Tyr Tyr Pro Glu Ala Leu Val Lys Tyr Ar - #g Leu His Ala Asn Gln          210              - #   215              - #   220                          - - Val Ser Ser Lys His Ser Val Arg Gln His Gl - #u Ile Ala Gln Gly Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gln Lys Thr Ala Arg Asn Asp Phe Leu Gln Se - #r Met Gly Phe Lys        Thr                                                                                             245  - #               250  - #               255             - - Arg Phe Asp Ser Leu Glu Tyr Arg Gln Thr Ly - #s Ala Ala Ala Tyr Glu                  260      - #           265      - #           270                  - - Leu Pro Glu Lys Asp Leu Pro Glu Glu Asp Ph - #e Glu Arg Ala Arg Arg              275          - #       280          - #       285                      - - Phe Leu Tyr Gln Cys Phe Lys Arg Thr Asp Th - #r Pro Pro Ser Gly Ala          290              - #   295              - #   300                          - - Trp Leu Asp Phe Ala Ala Asp Gly Arg Met Ar - #g Arg Leu Phe Thr Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Arg Gln Tyr Phe Gly Ile Leu Tyr Arg Leu Il - #e Lys Asn Arg Arg        Gln                                                                                             325  - #               330  - #               335             - - Ala Arg Ser Asp Ser Ala Gly Lys Glu Gln Gl - #u Ile                                  340      - #           345                                       __________________________________________________________________________

What is claimed is:
 1. A method of transferring at least two differentsaccharide units with a single enzyme comprising:(i) contacting a firstactivated saccharide unit with an acceptor moiety having a galactose atthe non-reducing terminus, in the presence of a polyglycosyltransferasethat catalyzes the linkages of said first activated saccharide unit withsaid acceptor moiety, to form a first saccharide composition; and (ii)contacting a second activated saccharide unit with a second saccharidecomposition comprising said first saccharide composition having agalactose at the non-reducing terminus, wherein said second activatedsaccharide unit is different from said first activated saccharide unit,in the presence of the polyglycosyltransferase, to form a preselectedthird saccharide composition.
 2. The method of claim 1, wherein saidfirst activated saccharide unit is N-acetylglucosamine orN-acetylgalactosamine.
 3. The method of claim 1, wherein thepolyglycosyltransferase is isolated from Neisseria.
 4. The method ofclaim 1, wherein the polyglycosyltransferase is isolated from Neisseriagonorrhoeae.
 5. A method of transferring at least two differentsaccharide units with a single enzyme comprising:(i) contacting a firstactivated saccharide unit with an acceptor moiety in the presence of apolyglycosyltransferase comprising the amino acid sequence of SEQ IDNO:8 that catalyzes the linkage of said first activated saccharide unitwith said acceptor moiety, to form a first saccharide composition; and(ii) contacting a second activated saccharide unit with a secondsaccharide composition comprising said first saccharide composition,wherein said second activated saccharide unit is different from saidfirst activated saccharide unit, in the presence of thepolyglycosyltransferase, to form a preselected third saccharidecomposition.
 6. The method of claim 5, wherein said first activatedsaccharide unit is N-acetylglucosamine or N-acetylgalactosamine.
 7. Themethod of claim 5, wherein said acceptor moiety and said secondsaccharide composition each have a galactose at the non-reducingterminus.
 8. A method of transferring at least two different saccharideunits with a single enzyme comprising:(i) contacting a first activatedsaccharide unit with an acceptor moiety in the presence of apolyglycosyltransferase encoded by a nucleic acid hybridizable tonucleotides 445-1488 of SEQ ID NO:1 that catalyzes the linkage of saidfirst activated saccharide unit with said acceptor moiety, to form afirst saccharide composition; and (ii) contacting a second activatedsaccharide unit with a second saccharide composition comprising saidfirst saccharide composition, wherein said second activated saccharideunit is different from said first activated saccharide unit, in thepresence of the polyglycosyltransferase, to form a preselected thirdsaccharide composition.
 9. The method of claim 8, wherein said firstactivated saccharide unit is N-acetylglucosamine orN-acetylgalactosamine.
 10. The method of claim 8, wherein said acceptormoiety and said second saccharide composition each have a galactose atthe non-reducing terminus.
 11. The method of claim 8, wherein saidnucleic acid is from a Neisseria strain selected from the groupconsisting of N. animalis (ATCC 19573), N. canis (ATCC 14687), N.cinerea (ATCC 14685), N. cuniculi (ATCC 14688), N. denitrificans (ATCC14686), N. elongata (ATCC 25295), N. elongata subsp. glycolytica (ATCC29315), N. elongata subsp. nitroreducens (ATCC 49377), N. flavescens(ATCC 13115), N. gonorrhoeae (ATCC 33084), N. lactamica (ATCC 23970), N.macaca (ATCC 33926), N. meningitidis, N. mucosa (ATCC 19695), N. mucosasubsp. heidelbergensis (ATCC 25998), N. polysaccharea (ATCC 43768), N.sicca (ATCC 29256) and N. subflava (ATCC 49275).
 12. The method of claim8, wherein said nucleic acid is from a microorganism selected from thegroup consisting of Branhamella catarrhalis, Haemophilus influenzae,Escherichia coli, Pseudomonas aeruginosa and Pseudomonas cepacia. 13.The method of claim 1, wherein said second activated saccharide unit isN-acetylglucosamine or N-acetylgalactosamine.
 14. The method of claim 5,wherein said second activated saccharide unit is N-acetylglucosamine orN-acetylgalactosamine.
 15. The method of claim 8, wherein said secondactivated saccharide unit is N-acetylglucosamine orN-acetylgalactosamine.